ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

· ELISA, stands for ‘’Enzyme-Linked Immunosorbent Assay”.

· Also known as EIA: Enzyme Immunoassay.

· It is commonly used technique to detect and quantify specific molecules, such as antigens or antibodies, in a sample.

· Small amount of specimen & a single dilution is required to perform ELISA test.

· Described by Eva Engvll and Peter Perlmann in 1971.

· It is widely employed in various fields, including clinical diagnostics, immunology, microbiology, and biomedical research.

ELISA PRINCIPLE

· ELISA principle is based on Antigen-Antibody interaction, the specific antibodies associated or bind to its target antigen. Only when the interaction take places the substrate can bind to the enzyme there by substrate conversion can be observed hence a positive result is obtained observed hence a positive result is obtained.

TYPES

· There are four major types of ELISA:

A) Direct ELISA: Detect primary antibody.

B) Indirect ELISA: Detect secondary antibody .

C) Sandwich ELISA: Two antibodies are employed, one to capture the target molecule and another to detect it, forming a "sandwich" complex.

D) Competitive ELISA: A labeled antigen competes with the unlabeled target molecule in the sample for binding to limited antibodies.

1. Direct ELISA:

· The target antigen is immobilized directly onto the solid surface (e.g., microplate).

· A labeled primary antibody, specific to the target antigen, is directly added and binds to the antigen.

· The labeled antibody generates a signal (e.g., color change or fluorescence) indicating the presence and quantity of the target antigen.

· Direct ELISA is simple and straightforward but may have limitations in sensitivity.

2. Indirect ELISA:

· The target antigen is immobilized onto the solid surface.

· A primary antibody, specific to the target antigen, is added and binds to the antigen

· A secondary antibody, labeled with an enzyme or fluorophore, is introduced and binds to the primary antibody.

· The labeled secondary antibody amplifies the signal, enhancing sensitivity compared to direct ELISA.

· Indirect ELISA allows for signal amplification and detection of multiple primary antibodies using a single labeled secondary antibody.

3. Sandwich ELISA:

· A capture antibody is immobilized onto the solid surface, specifically binding to the target antigen.

· The sample containing the target antigen is added, and the antigen binds to the capture antibody.

· A detection antibody, specific to a different epitope of the antigen, is added and binds to the antigen, forming a "sandwich" complex.

· A labeled secondary antibody, recognizing the detection antibody, is introduced and binds to the detection antibody.

· Sandwich ELISA provides high specificity and sensitivity, as it involves the use of multiple antibodies to capture and detect the target antigen.

4. Competitive ELISA:

· The solid surface is coated with specific antibodies.

· A labeled antigen and an unlabeled antigen (competitor) are simultaneously added to the surface.

· The labeled and unlabeled antigens compete for binding to the limited number of antibodies.

· The signal generated is inversely proportional to the amount of unlabeled antigen in the sample.

· Competitive ELISA is commonly used for detecting small molecules or haptens.

ELISA PROCEDURE

· ELISA test procedure including following steps :

1. Coating:

· Coat the solid surface (microplate wells) with the antigen or antibody of interest.

· Incubate the coated plate at an appropriate temperature to allow the molecules to bind to the surface

2. Blocking:

· Block the remaining uncoated surfaces on the plate with a blocking agent (e.g.serum).

· Incubate the plate to prevent non-specific binding.

3. Sample and Standards:

· Add the samples (patient samples or experimental samples) and a series of known concentration standards to separate wells.

· Incubate the plate to allow the molecules in the samples to bind to the coated surface.

4. Washing:

· Wash the plate multiple times with a washing buffer to remove unbound substances and reduce background noise.

· Proper washing is essential to maintain assay specificity and reduce false positives.

5. Detection:

· Add a detection antibody specific to the target molecule of interest.

· The detection antibody is often labeled or conjugated with an enzyme for signal generation.

· Incubate the plate to allow the detection antibody to bind to the target molecule or antigen.

6. Washing:

· Wash the plate again to remove unbound detection antibodies.

7. Substrate Addition:

· Add a substrate solution that reacts with the enzyme label on the detection antibody.

· The substrate reaction generates a detectable signal, such as a color change or fluorescence.

8. Signal Measurement:

· Measure the signal using a spectrophotometer or a specialized ELISA plate reader.

· Determine the absorbance or fluorescence intensity in each well, which is proportional to the amount of target molecule present.

9. Data Analysis:

· Compare the signal of the samples to the standard curve generated from the known concentration standards.

· Quantify the concentration of the target molecule in the samples based on the standard curve.

· Calculate and report the results.

RESULT INTERPRETATION IN ELISA

Result Interpretation:

· For qualitative assays, such as presence/absence determination, a threshold value is set, and samples with signal above the threshold are considered positive.

· For quantitative assays, the concentration of the target molecule is reported for each sample.

APPLICATION OF ELISA

1. Medical Diagnostics:

· ELISA is widely used in medical diagnostics to detect and quantify various disease markers, including viral infections, autoimmune diseases, hormonal imbalances, and cancer biomarkers.

· It is employed for screening, diagnosis, prognosis, and monitoring of diseases.

· Example ELISA can detect antibodies against HIV, hepatitis viruses, or detect specific antigens or antibodies related to autoimmune disorders.

2. Infectious Disease Testing:

· ELISA is utilized for the detection of infectious agents, such as viruses, bacteria, and parasites, in patient samples.

· It is used in the diagnosis of infectious diseases like HIV, hepatitis, Lyme disease, and sexually transmitted infections (STIs).

3. Allergen Testing:

· ELISA plays a crucial role in identifying allergens in food products and environmental samples.

· It is used to detect allergens like peanuts, milk, eggs, gluten, and other common allergenic substances.

· ELISA-based allergen testing helps ensure food safety and labeling compliance for individuals with food allergies.

4. Research and Biotechnology:

· ELISA is extensively utilized in research laboratories for studying protein-protein interactions, protein quantification, and biomarker discovery.

· ELISA is employed in fields like immunology, molecular biology and drug development.

5. Veterinary Diagnostics:

· ELISA is used in veterinary diagnostics for the detection of infectious agents and specific antibodies in animals.

6. Food Safety and Quality Control:

· ELISA is employed in the food industry to detect contaminants, toxins, and pathogens in food products.

· It helps ensure the safety and quality of food by detecting substances like pesticides, mycotoxins and microbial contaminants.

ADVANTAGE OF ELISA

1. Sensitivity: ELISA is highly sensitive, capable of detecting very low concentrations of target molecules.

2. Specificity: ELISA offers high specificity, it minimize false-positive and false-negative results, leading to more accurate and reliable diagnostic outcomes.

3. Quantitative Results: ELISA can provide quantitative measurements of target molecules.

4. Relatively Simple Procedure: ELISA has a well-established and standardized procedure that is relatively straight-forward to perform. Many commercially available ELISA kits provide pre-coated plates and optimized reagents, simplifying the process and reducing variability.

5. Cost-Effectiveness: ELISA is generally a cost-effective assay, especially when considering its sensitivity and specificity.

6. Stability and Long Shelf Life: ELISA reagents, such as coated plates and enzyme-conjugated antibodies, can have long shelf lives when stored properly.

7. Wide Range of Applications: ELISA has diverse applications in medical diagnostics, research, veterinary medicine, food safety, and environmental monitoring.

DISADVANTAGE OF ELISA

1. Limited Multiplexing: ELISA typically allows the detection of only one target molecule at a time in a single assay.

2. Time-Consuming: ELISA assays often require several hours to complete, including sample preparation, incubation, washing steps, and signal development..

3. Limited Dynamic Range: ELISA has a finite dynamic range, which means that extremely high or low concentrations of target molecules may fall outside the quantifiable range.

4. False Positives and Negatives: ELISA can still be susceptible to false-positive or false-negative results. Factors such as cross-reactivity, sample matrix interference, or non-specific binding can lead to inaccuracies in the results.

5. Sample Complexity: Complex matrices, such as serum, plasma, or tissue extracts, may contain various interfering substances that can affect assay performance.

6. Limited Shelf Life of Opened Kits: Once opened, ELISA kits often have limited stability and shelf life for the reagents. Proper storage conditions and careful management of kit components are necessary to ensure accurate and reliable results.

7. Operator Skill and Training: ELISA requires skilled operators who are familiar with the assay principles, techniques, and interpretation of results.

LIMITATION OF ELISA

1. Sensitivity Limit: ELISA has lower limit of sensitivity, which not detect very low concentrations of target molecules.

2. Cross-Reactivity: where antibodies may bind to similar molecules or structures other than the intended target. This can lead to false-positive results.

3. Limited Dynamic Range: ELISA has a finite dynamic range, which means that very high or very low concentrations of the target molecule may fall outside the quantifiable range. Dilutions or alternative assay methods may be necessary to accurately measure samples with wide concentration ranges.

4. Subjectivity in Interpretation: The interpretation of ELISA results can involve subjective decisions, such as setting cutoff values or determining the presence of a positive or negative signal.

5. Time-Consuming: ELISA assays typically require several hours to complete, including incubation and washing steps. This longer assay duration can be a limitation when rapid results are needed, such as in emergency or time-sensitive situations.

Cost: ELISA assays can be cost-effective for routine testing, but they may become expensive when large-scale testing or multiple analyte detection is required. Costs can include reagents, consumables, equipment, and specialized kits.

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